P1567: ALK2 INHIBITION LOWERED HEPCIDIN AND LIBERATED SPLEEN IRON FOR ERYTHROPOIESIS IN ANEMIA OF INFLAMMATION
Authors: C.-Y. Wang, N. Medeiros, E. Melgar-Bermudez, T. Nurse, E. Lema, C. Tseng, F. Fisher, J. Seehra, J. Lachey
Journal: HemaSphere (2022)

Background: Anemia of inflammation (AI) caused by persistent inflammation is common in chronic disease. Inflammatory cytokines, including interleukin 6 (IL-6), induce expression of hepcidin, an iron homeostasis regulator, which retains tissue iron and limits circulating iron, leading to anemia. Iron and inflammation regulate hepcidin through the bone morphogenetic protein (BMP)-SMAD and IL6-STAT3 pathways, respectively. BMP2 and 6 bind BMP receptors, including ALK2, to phosphorylate SMAD1/5/9, while IL-6 signals through STAT3. Activated SMAD1/5/9 or STAT3 bind independently to hepcidin promoter regions to initiate transcription. Evidence suggests that targeting BMP-SMAD to reduce hepcidin may ameliorate anemia in AI. We developed two investigational neutralizing antibodies against ALK2, KTI-m216 (m216) and KTI-m218 (m218), with unique variable regions but observed to have similar affinity for ALK2 and effect on hepcidin suppression in mice and monkeys. Aims: We investigated the mechanism of ALK2 inhibition-mediated hepcidin suppression in acute inflammation and chronic AI mouse models. Methods: C57BL/6 mice were dosed with 3 mg/kg m216 or isotype control (Ctrl) for 1 h, followed by 1 mg/kg lipopolysaccharide (LPS) or PBS for 6 h. For an AI model, C57BL/6 mice were fed a control or 0.2% adenine diet with 40 ppm iron for 6-7 weeks to induce chronic kidney disease (CKD), followed by 3 mg/kg m218 (CKD-m218) or isotype (CKD-Ctrl) twice weekly for 3 more weeks. In a separate experiment, CKD mice were switched to a similar adenine diet with low 3 ppm iron when receiving m218 or Ctrl for 9 days. Results: Serum IL-6 was similarly induced in the Ctrl-LPS and m216-LPS mice, compared to respective PBS controls. Consistent with previous findings, m216 reduced serum hepcidin in PBS-treated mice (Ctrl-PBS 509 ng/ml; m216-PBS 78 ng/ml). While LPS induced serum hepcidin in Ctrl-LPS (801 ng/ml) and m216-LPS (247 ng/ml) mice, compared to respective PBS controls, absolute hepcidin was lower in the m216-LPS mice, suggesting that m216 reduced serum hepcidin by lowering basal transcription but not preventing LPS induction. To examine potential crosstalk between the two pathways, phosphorylation of liver SMAD5 (PSMAD5) and STAT3 (PSTAT3) were examined by Immunoblot. PSTAT3 was increased similarly in Ctrl-LPS and m216-LPS mice, compared to respective PBS controls, which is consistent with the similar serum IL-6 in these mice. PSMAD5 was not induced by LPS but, as expected, was decreased by m216 compared to Ctrl-PBS mice. These data support that BMP regulation of hepcidin may be distinct from regulation by IL-6. Importantly, ALK2 inhibition alone was sufficient for hepcidin reduction in inflammation. CKD resulted in increased serum hepcidin, decreased serum iron and increased liver and spleen iron retention, compared to mice on a control diet. CKD-m218 mice had an 82% reduction in serum hepcidin and a 73% increase in serum iron after 3 weeks of treatment compared to CKD-Ctrl mice. Additionally, CKD-m218 mice had a 47% reduction in spleen iron and a 1.4 g/dL increase in hemoglobin compared to CKD-Ctrl mice. Similar responses were observed in CKD-m218 mice fed a low iron diet, suggesting that the increased iron in CKD-m218 mice was mostly from the spleen rather than diet. Summary/Conclusion: These data suggest that IL-6-induced hepcidin is largely independent of ALK2, but ALK2 inhibition may be sufficient to result in a net hepcidin reduction and improved erythropoiesis. Data also suggest that, in AI with adequate iron stores, ALK2 inhibition potentially acts by liberating iron from the recycling pathway.